Thymic stromal lymphopoietin (TSLP) exerts a marked influence on the polarization of dendritic cells to drive T helper (Th) 2 cytokine production, and has been linked to allergic airway diseases. Although TSLP is produced by airway epithelium, TSLP production in laryngeal arytenoid fibroblasts remains largely unexplored. We examined the effect of Toll-like receptor (TLR) ligands and the cross-talk that occurs among different TLR ligands on TSLP production in arytenoid fibroblasts. Since mRNA of TLR 2, 3, 4, and 9 has been found to be expressed in arytenoid fibroblasts, we examined the effect on its production of TLR ligands. TSLP production by arytenoid fibroblasts was strongly induced in the presence of polyinosinic-polycytidylic acid (poly(I:C)), a ligand of TLR3. Its production was synergistically induced in the presence of IL-4, to a level more than 100 times higher than that observed in the absence of poly(I:C) or IL-4. We also revealed that B type DNA containing CpG motifs (CpG-DNA) coding for a TLR9 ligand markedly suppressed both poly(I:C)-induced and poly(I:C)-plus-IL-4-induced TSLP production. B type CpG-DNA decreased the poly(I:C)-induced phosphorylation of c-Jun N-terminal kinase (JNK), and pre-incubation with SP600125 (inhibitor of JNK) reduced the poly(I:C)-induced TSLP-production. These results indicate that human arytenoid fibroblasts strongly induce TSLP production with stimulation by double-stranded RNA (dsRNA), which can be inhibited by CpG-DNA and participate in immune allergic responses. (C) 2011 Elsevier Ltd. All rights reserved.